The overall aim of this project involves the use of nuclear magnetic resonance (NMR) spectroscopy to characterize biological macromolecules and their interaction with compounds of environmental or pharmacological concern. The formation of protein adducts by environmental agents involves complex chemical/biochemical/structural interactions which are, at best, incompletely understood. During the past year, NMR studies of the interaction of [acetyl-1-13C]aspirin with the model protein ubiquitin were performed. These studies provide the first example in which the protein modification process could be followed for individual residues in real time. Adduct resonances were assigned based on structural data and utilizing lysine -< arginine mutants prepared by our collaborator, Prof. Arthur Haas. Modification of lysine residues correlate fairly well with their surface accessibility, and less closely with lysine pK values. Studies of reductively methylated ubiquitin indicate the presence of long range interactions and exhibit interesting pH dependent effects. Studies of human hemoglobin modifed with [acetyl-1-13C]aspirin have been performed because hemoglobin acetylation has been proposed as a useful strategy for reducing the tendency of sickle cell hemoglobin (HbS) to aggregate. Aspirin was one of the earliest agents studied for this purpose, and is known to modify residues in the diphosphoglycerate binding pocket. We have utilized ligands such as inositol hexaphosphate and the paramagnetic shifts observed in cyanomethemoglobin in order to obtain assignment data for the acetyl resonances. In general, these studies indicate that the acetylation of hemoglobin by aspirin, as well as by some more recently developed acetylating agents, is less specific than had previously been suggested.